MIC 206: Lab 14, 15 - Acid fast, Capsule Stainings

What does simple staining do

Allows one to identify cell morphology and arrangement

What does gram staining do

Allows one to differentiate 90% of bacterial species into 2 large groups (gram positive and gram negative) based on chemical and physical properties of their cell walls (peptidoglycan)

What is acid-fast bacteria

Unique cell wall which contains mycolic acid

What is mycolic acid (acid-fast staining)

Composed of fatty acids, waxes, and complex lipids that make up the cell walls
Ex: helps protect against certain antibiotics/antimicrobials

What is the name of the example genus that is a known acid-fast organisms that is responsible for diseases like Tuberculosis and Leprosy (acid-fast staining)

Mycobacterium (mycobacterium tuberculosis and mycobacterium leprosy)

What are the steps of an acid-fast stain - Ziehl Neelsen (acid-fast staining)

1. Smear prep and heat fix

2. Flood slide with Carbolfuchsion (primary stain) for 8 minutes over steam

3. Wash with water

4. Carefully decolorize with acid-alcohol (decolorizing agent) → acid-fast cells resist decolorization by an acid alcohol

5. Wash with water

6. Flood with methylene blue (counterstain) for 60 seconds

7. Wash with water

8. Blot with absorbent paper

9. View with oil objective

What is the primary stain for acid-fast stain and how does it relate to steam (acid-fast staining)

Carbolfuchsion: is a lipid soluble stain that penetrates the waxy cell wall.
The steam melts the wax in the cell wall to allow the stain to move into the cell

What is the counterstain for acid-fast stain (acid-fast staining)

Methylene blue

What color are acid-fast and nonacid fast cells

Acid-fast cells are reddish/pink
Nonacid-fast cells are blue

What type of stain is this

Acid-fast stain

What is the capsule of a cell (capsule stain)

Water-soluble, preventing dye from adhering to them.
Composed of polysaccharides/polypeptides

What is the function of a capsule

Increase virulence by protecting the bacteria against phagocytosis and antibodies.
It helps the bacteria attach to surfaces

What is used to stain the background for a capsule (capsule stain)

Negative stain stains the background which surrounds unstained microbes

What is the negative dye used in capsule stain (capsule stain)

Nigrosin, which is negatively charged and is repelled by the negative charge (-) of the bacteria.
It is an acidic dye because it gives up a hydrogen ion

What is the positively charged dye used in capsule stain (capsule stain)

Crystal violet, which is positively charged and stains the bacteria cells.
It is a basic dye that stains purple on a dark background

How do you know if a bacteria has a capsule (capsule stain)

Purple cells are surrounded by a clear, colorless zone which is the capsule

How do you do a capsule stain negative (capsule stain)

1. Apply a line of bacteria to 1 end of the slide

2. Add 1 drop of Nigrosin to the same end of slide and mix with loop

3. Take a second clean slide and place it on the surface of the first slide at the nigrosin drop site

4. Hold a clean slide at 45 degree angle and draw it back to the other side of the slide, spreading the stain and bacteria throughout the stained slide

5. Air dry, DO NOT heat fix

6. Flood slide with crystal violet for 1 minute

7. Wash with water

8. Gently blot with absorbent paper

9. View with oil objective

What stains are used in a capsule stain Anthony method versus capsule stain negative method (capsule stain)

Anthony method: Crystal violet (+ basic dye to stain bacterial cells) & Copper sulfate (decolorizing agent removing the crystal violet and absorbing the cooper sulfate which is blue)
Negative method: Nigrosin and Crystal violet

What will the Anthony method of capsule stain look like (capsule stain)

Purple cells surrounded by a blueish zone (which is the capsule)

What is the procedures to a capsule stain Anthony method (capsule stain)

1. Apply 3 loopfuls of bacteria to one end of the slide. If from plate media, apply a line of bacteria and add a drop of crystal violet

2. Take a second clean slide and place it on the surface of the first slide at the crystal violet drop site

3. Hold clean slide at 45 degree angle and draw it back to the other side of the slide, spreading the stain and bacteria throughout the stained slide

4. Allow slide to air dry for 7 minutes (do not heat fix)

5. Wash the slide with Copper Sulfate for 2 seconds

6. Gently blob with absorbent paper

7. View with oil objective